The diagnosis/prognosis of a possible cancer typically includes the removal of a cell sample, such as a tissue mass, from the patient. Although an attending physician may have good intuition regarding the patient's diagnosis/prognosis, confirmation of the diagnosis with a histological examination of the cell sample removed from the patient is necessary. The histological examination entails cell staining procedures which allow the morphological features of the cells to be seen relatively easily in a light microscope. A pathologist, after having examined the stained cell sample, makes a qualitative determination of the state of the tissue and reaches a conclusion regarding the prognosis for the patient. While this diagnostic method has a long history, it is somewhat lacking in scientific rigor since it is heavily reliant on the subjective judgment of the pathologist and it is extremely time consuming.
Typically, the cell samples are embedded in a paraffin block from which tissue sections are cut by a microtome instrument. The optical evaluation of such cell samples, particularly those taken from microtomed tissue sections, is a difficult procedure. The optical field presented to an evaluator is a disordered collection of cell objects, some on top of one another and others being only fragments of whole cell objects. The optical field shows only boundaries of two-dimensional optical entities filled with varying levels of contrast. Some of the overlapped cell objects appear to be large and/or dense single cell objects and some of the cell object fragments appear to have sufficient size to be whole cell objects. Faced with this random cluster of images, the evaluator's difficult and time-consuming task is the selection of single whole cell objects which can accurately represent the cell sample and the classification of those selected objects into categories which classification aids in the final diagnosis/prognosis.
It is well known that the DNA content of cell objects can provide valuable information in cancer diagnosis. Systems have been developed which utilize the DNA content of cell objects to improve histological examination. In U.S. Pat. No. 4,741,043 to Bacus for Method and Apparatus for Image Analyses of Biological Specimens, an automated method and a system for measuring the DNA of cells are disclosed which employ differential staining of the DNA in cell nuclei with a Feulgen stain and image processing. After staining, optical fields of the cell sample are presented to an evaluator who selects objects for analysis and categorizes the selected objects. Certain attributes including the DNA mass of the operator selected cell objects are then measured and used to produce reports such as DNA histograms.
The arrangement and method of Bacus U.S. Pat. No. 4,741,043 have been well received both for the reports generated and for the improvements in the use of operator time. The operator, however, must still select relevant cell objects from the optical field presented and classify the selected cell objects into classes before machine measurement of attributes occurs. Such selection and classification is especially tedious in cut tissue section specimens, in contrast to whole cell preparations. It requires the thoughtful review of each object in the random cluster of images of an observed field. Further, the only input information available for such review is the varying contrast levels presented by the visual image. When the operator must evaluate cell samples for a long period of time, as is the case in some pathology laboratories, concentration by the operator and accuracy of the decisions made, may be affected.
This need for a more automated method and arrangement for use with a DNA analysis apparatus, which selects whole, single cell objects and classifies each selected cell object as being in a particular one of a plurality of diagnostic aiding categories as well as in particular regions of the DNA distribution has been meet by the arrangement and method disclosed in co-pending patent application Ser. No. 07/595,117, filed Oct. 10, 1990, U.S. Pat. No. 5,235,552. This automatic selection and classification of cell objects speeds analysis and reduces the tedium of the operator. Also, pre-selection and classification by the apparatus permits the operator to concentrate his or her efforts on the difficult and subtle analysis of the preselected cell objects which are likely to be representative of the sample. With this system, the measured DNA mass of such fragments can be corrected (increased) to reflect the DNA mass of their source whole cell object as suggested by McCready et al. in an article published in Analytical Quantitative Cytology, Vol. 5, No. 5, June 1983, pp. 117-123, entitled "An Analysis of DNA Cytophotometry on Tissue Sections in a Rat Liver Model," the disclosure of which is hereby incorporated by reference in its entirety. After the automatic measurement of cell object attributes, cell object fragments which are likely to have analysis value are identified from their measured attributes and the cell object attributes are corrected to reflect the attributes of the whole cell objects from which the fragment was sectioned.
The valuable cell object fragments are then identified by comparing the measured cell object area with a threshold value determined from the thickness of a tissue section from which the sample was taken. If the measured cell object area is larger than the threshold, the cell object is identified for correction since it is too large to be entirely included within the tissue section. A correction value is then determined from the measured area and the tissue section thickness and used to increase the measured DNA mass of the identified cell object fragment.
The correction value C is determined from the equation: ##EQU1## where T is the tissue section thickness and R equals the square root of the measured area divided by .pi.. The formula yields a correction value between 0 and 1 for identified cell objects, which value is divided into the measured DNA mass of the cell object to increase the DNA mass to a corrected value representing the whole cell object.
A problem and limitation in the evaluation of the cell objects and their accurate reporting remains as a result of variations in the thickness of the tissue section used in determining the variation constant C. Normally, the microtome instrument is set, e.g., at 5.mu., and this thickness setting is used by the particular operator and/or the particular lab. However, the act of slicing the tissue section results in variations in the thickness of the tissue section as suggested by Allison et al. in an article published in the Journal of Microscopy, Vol. 159, Pt. 2, August 1990, pp. 203-210, entitled "Measuring the forces acting during microtomy by the use of load cells." Thus, the actual thickness may vary depending upon the conditions present during cutting of the paraffin block and the microtome setting may not accurately reflect the actual thickness of the tissue section from which the sample was taken.
Thus, a need exists for methods and apparatus for measuring the thickness of tissue sections to compensate for variations from the microtome setting, and especially a method that can be used in conjunction with an analysis of DNA, thereby, improving the accuracy and reliability of the image analysis systems used to identify the cell object fragments likely to possess analysis value and properly correct their measured attributes to reflect what those attributes would have been, had the identified cell object not been fragmented. In addition, there has been a need for such methods and apparatus to measure the thickness of the tissue section for comparison with the nominal thickness determined from the microtome setting to calibrate the microtome instrument and to provide some degree of quality control in connection with the microtome instrument.